Long before 1936, Van Klobusitzky et al., the scholars of Austria, purified and refined an enzymatic haemostatic, batroxobin, from the venom of Bothrops atrox. The initial translation of the batroxobin in China was “baqimei”. Batroxobin belongs to the serine proteases family. Its physiological function and molecular size are similar to those of thrombin. Therefore, it was called as “xueningmei” later. However, people at home and broad defined batroxobin with different meanings in different periods along with the development of research. There are five subspecies for Brazilian lancehead snake (Bothrops atrox). The batroxobin obtained from certain subspecies exhibits the hemostatic efficacy. The batroxobin obtained from other subspecies, however, exhibits the function of removing fibrinogen. As a result, the difference in the species of snake results in that the proteins generally called as “baqimei” in fact exhibit completely different biological and chemical essence. Some of them possess hemostatic efficacy as their main function, while the other possess the degradation of fibrinogen as their main function. Further investigation and research are needed to determine whether or not said difference in chemical essence is derived from the difference in the amino acid sequence or from the different in the protein modification, such as glycoconjugates and the like.
Currently, there are some patents (U.S. Pat. Nos. 5,595,974, 5,869,044, 6,106,830, 6,399,576, 6,416,717; Eur. Pat. Nos: 0984279, 0826374, 0750912, 0719791) and investigation reports relating to the use of batroxobin that is extracted and prepared only from the snake venom in treating many clinical indications, such as myocardial infarction, senile dementia, stroke, sudden deafness and the like. No researches are reported about the animal tests and clinical investigation of the recombinant batroxobin. The batroxobin biologically extracted from the snake venom is mainly obtained from the venom of Bothrops moojeni, Bothrops atrox. However, the content is low and it is difficult to obtain the starting material and the purified natural batroxobin. Generally, there are a few of snake toxins and many unknown impurities retained in the final product. This raises the potential risk for clinical use. The theoretical molecular weight of the highly purified natural batroxobin should also be 25.6 kDa. However, in fact, the actual molecular weight are 37-43 kDa because the protein has experienced a glycosylation modification when it is secreted and expressed in the cells of poison gland of the viper. The alteration of molecular weight may result from the degree of glycosylation modification or the loss of the sugar chain due to different purification processes, and the like. Since the source of the starting material, i.e., the snake venom, is influenced by the scale of the cultivated snakes and the change of four seasons, it is difficult to control the quality of natural batroxobin extracted and prepared from the snake venom and the specific activity thereof is very unstable.
The molecular biology study on batroxobin, a protein obtained from the snake venom, advanced slowly until in 1987 and 1988, the investigators in Japan finished the sequencing work for the cDNA and genomic DNA of batroxobin gene. In 1991, the investigators in Fujisawa Pharmaceutical (Japan) used a fusion expression system in E. coli to express the component as inclusion body via the gene recombinant method. And then they obtained batroxobin, which they claimed had a biological activity, by electrophoresis and cleaving the fusion protein with thrombin and they filed an patent application (Pat. No. JP2124092, 1990). It is always technically difficult to produce the protein enriched in disulfide bonds via the genetic engineering means, especially for the serine proteolytic enzymes containing many pairs of disulfide bond. This is because the disulfide bonds may mismatch in high rate and the products obtained in prokaryotic expression system are almost in the form of inclusion bodies. Although the Fujisawa Pharmaceutical Co., Ltd in Japan reported that the target protein having activity could be obtained via denaturing and re-naturing the inclusion body, the specific activity of the protein is low and the reproducibility is poor. And until now it is not found that the recombinant batroxobin product of that company has been produced and marketed.
Therefore, there still is an urgent need in the art to provide a recombinant batroxobin product with high activity, the production of which is not limited by the seasons or the production scale is readily controlled, and which has a high rate in the correct matching of the disulfide bond.